Parathyroid hormone regulates calcium and bone metabolism by controlling critical functions of target cells in bone and kidney. These actions of the hormone were formerly attributed entirely to stimulation of adenylate cyclase, but functional analysis of the recently cloned PTH/PTHrp receptor has confirmed that this single receptor can transduce multiple intracellular signals, including increases in cyclic AMP, diacylglycerol, inositol polyphosphates and cytosolic free calcium. The ultimate functional correlates of this signalling diversity are poorly understood, however, because the links between individual second messengers and PTH regulation of specific distal biologic responses, such as growth, gene transcription, enzyme activity and ion transport, have not been clearly established. The importance of interactions between these various second messengers; their modulation by other hormones, growth factors or cytokines; and the possible influence of different schedules of hormone administration upon the patterns of hormonal responsiveness also remain unsettled. Recent advances in the development of signal-specific PTH analogs, coupled with the availability of native and mutant PTH receptor cDNAs, have inspired new experimental approaches to these issues. This Subproject will address two major unresolved questions concerning the cellular actions of PTH: (1) how do individual second messengers elicited by the hormone influence specific distal functions of target cells - i.e. do different signals principally regulate distinct subsets of PTH responses in these cells? and (2) how are PTH responses in target cells influenced by the temporal pattern of hormone exposure - are specific signalling mechanisms or biologic responses regulated differently by continuous vs. intermittent (or pulsatile) PTH administration? by using mutant PTH ligands, mutant PTH receptors and mutant renal and osteoblastic target cells to selectively activate, or block, specific PTH signalling events in vitro, the links between generation of individual second messengers and specific distal cellular effects of the hormone will be defined. In the case of osteoblasts, particular emphasis will be placed upon distinguishing those signals that mediate "anabolic" vs. "catabolic" effects of PTH on bone. To gain insight into the mechanisms whereby intermittent and continuous PTH administration in vivo lead to dramatically different effects upon bone mass perifusion techniques will be used to compare the effects of continuous vs. intermittent or pulsatile PTH exposure upon cells expressing either normal PTH/PTHrp receptors or mutant receptors that cannot transduce specific messenger signals. Confirmation of key findings in transformed osteosarcoma-cell models will be sought using primary bone cells from normal mice or from gene-ablated mice that are homozygous for PTH receptor deficiency and in which mutant PTH receptors have been "transplaced" in vivo or in vitro. By defining the functional consequences of individual signals transduced by PTH receptors in target cells, these studies will provide the information needed for rational design of signal-specific PTH analogs with novel spectra of action in kidney and bone. They may also suggest new pharmacokinetic approaches that, together with such novel PTH analogs, could ultimately enable highly selective systemic control of bone turnover and mineral metabolism.